Cannabis sativa and C. indica) is one of the most common illegal drugs used by drug abusers. Indian cannabis contains around 70 alkaloids, and delta-9-tetrahydrocannabinol (delta-9-THC) is the most psychoactive substance. Animal intoxications occur rarely and are mostly accidental. According to the US Animal Poison Control Center, cannabis intoxication mostly affects dogs (96%). The most common cause of such intoxication is unintentional ingestion of a cannabis product, but it may also occur after the exposure to marijuana smoke.Cannabis from hemp  at 5.5 ng/mL, 11-hydroxy-delta-9-THC at 1.2 ng/mL, and 11-carboxy-delta-9-THC at 13.8 ng/mL. The cat was given an isotonic solution of NaCl 2.5 and 2.5% glucose at a dose of 40 mL/kg/day parenterally and was hospitalised. After complete recovery, the cat was returned to it’s owner and future isolation of the animal from marijuana smoke was advised.This is the first case of a delta-9-tetrahydrocannabinol intoxication in a cat with both description of the clinical findings and the blood concentration of delta-9-THC and its main metabolites. Cannabis sativa and C. indica) is one of the most common illegal drugs used by drug abusers. Cannabis resin and oil are extracted from hemp plants. Indian cannabis contains approximately 70 alkaloids with delta-9-tetrahydrocannabinol (THC) being the most psychoactive compound. The concentration of delta-9-THC in the plant depends on the part of the plant, with the highest concentration in the feather crown and on the cultivation conditions. The concentration of delta-9-THC in the desiccated plant amounts to 12%, but when smoked not more than around 20% is transmitted into the smoke [Cannabis from hemp (https://www.aspca.org/), cannabis intoxication mostly commonly affects dogs (96%) and more rarely cats (3%) but may also occur sporadically in other species. The most common cause of intoxication is an erroneous ingestion of a cannabis product, as well as secondary intoxication through exposure to marijuana smoke. The most popular form of cannabis, which is also eaten by pets is “marijuana butter” or so called “hash cookies”, acquired by placing parts of the plant in butter. As a result, the fat-soluble THC is extracted from the plant into the fat present in butter, reaching high concentrations [Animal intoxication occurs rarely and is most often accidental. Episodically, intentional exposure of an animal to marijuana may occur. According to the US Animal Poison Control Center .In the follow-up interview it turned out, that the aggressive behaviour commenced after longer absences of the owner of at least a couple of days, when the owner’s partner looked after the cat. The examining veterinarian asked the owner to contact her partner in order to identify possible causes of the medical condition of the cat. It turned out that the partner was marijuana-dependent and he admitted to have smoked marijuana while the cat’s owner was absent and to have exhaled marijuana smoke directly in the cat’s face “as a joke”.The cat was given an isotonic solution of NaCl 2.5 and 2.5% glucose  parenterally at a dose of 40 mL/kg/day for 2 days and was hospitalised in order to monitor the condition. During the 48-h hospitalisation, there were two periods of agitation and aggression. Sedatives were not applied as the attacks lasted less than a few minutes.The blood sample underwent toxicological analysis. A screening enzyme-linked immunoassay analysis (ELISA) was used to screen for opiates, cannabinols, benzodiazepines, amphetamines and their derivatives as well as cocaine. A positive result was observed for cannabinols. Hence, further verification and quantitative tests were conducted with the use of gas chromatography tandem mass spectrometry (GC–MS) . The blood tests revealed the presence of delta-9-THC at 5.5 ng/mL, 11-hydroxy-delta-9-THC at a concentration of 1.2 ng/mL, and 11-carboxy-delta-9-THC at a concentration of 13.8 ng/mL.Taking into account the clinical signs as well as the result of the toxicological tests, marijuana intoxication was diagnosed as the cause of the cat’s disease. The animal was returned to its owner and future isolation of the animal from marijuana smoke was advised.The toxicokinetics of delta-9-THC in cats has not been reported yet. In humans and laboratory animals, the metabolism of delta-9-THC depends mostly on the exposure route. Following inhalation, the concentration of THC rapidly increases in the blood, reaching its maximum concentration within a couple of minutes. When marijuana is ingested, the absorption of delta-9-THC is slower, and maximum blood concentration peak occurs 30–60 min after consumption. Delta-9-THC is rapidly metabolized by the liver, changed to 11-hydroxy-delta-9-THC and then oxidized to 11-carboxy-delta-9-THC. There are other delta-9-THC metabolites, but THC-OH and THC-COOH are the key cannabinoids used in the diagnosis of marijuana intoxication in humans [Although studies have been conducted on neurological diseases , 4, inhiThe minimal toxic or lethal dose of delta-9-THC for a cat is unknown. In dogs, this dose must exceed 3 g/kg when administered orally  which isThe clinical signs of an intoxication with delta-9-THC in cats differ from those in humans, and include disorders of consciousness, possibly leading to a coma, convulsions, ataxia, depression or agitation, anxiety, vocalization, hypersalivation, diarrhoea and vomiting, bradycardia or tachycardia, hypothermia and mydriasis .The clinical signs of a marijuana intoxication in dogs have been reported by Janczyk et al.  but therCats may be prone to develop marijuana intoxication or cancers of the mouth and lymph nodes because of second-hand tobacco exposure . When caThe number of countries allowing legal use of marijuana for medical purposes has increased. In addition, some pet owners find marijuana to be a safe stimulant and smoke it for recreation at home in the presence of their pets. This case shows that the symptoms of delta-9-THC intoxication are unspecific and may resemble intoxication with other substances. It is advisable that the attending veterinarian consider toxicological tests and consider various causes of neurological dysfunction, outside standard causes, in order to diagnose and treat such cases.This is the first laboratory confirmed published case of the clinical signs of a cat being intoxicated with delta-9-THC. Screening tests for use in humans proved useful for cats as well.


Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that can lead to cirrhosis, liver transplant, and even hepatocellular carcinoma. While liver biopsy remains the reference standard for disease diagnosis, analytical and clinical development of non-invasive soluble biomarkers of NASH are of great importance to advance the field. To this end, we performed analytical and clinical validation on a series of pro-inflammatory cytokines and chemokines implicated hepatic inflammation; IL-6, CRP, TNFα, MCP-1, MIP-1β, eotaxin, VCAM-1. Biomarker assays were validated for accuracy and precision. Clinical performance was evaluated in a random sample of 52 patients with biopsy-proven NAFLD/NASH. Patients were categorized into three groups according to their fibrosis stage; advanced (F3-F4), mild (F1-2) and no (F0) fibrosis. Serum IL-6 was increased in patients with advanced fibrosis  compared to patients with mild or no fibrosis respectively. While, there was no significant difference noted in CRP, TNFα, MCP-1, MIP-1β, eotaxin among the three groups, VCAM-1 levels were increased by 55% (p<0.01) and 40% (p<0.05) in the advanced cohort compared to the mild and no fibrosis groups respectively. VCAM-1 also displayed good clinical performance as a biomarker of advanced fibrosis with an area under the receiver operating curve of 0.87. The VCAM-1 assay demonstrated robust accuracy and precision, and VCAM-1 outperformed IL-6, CRP, TNFα, and the chemokines MCP-1, MIP-1β, and eotaxin as a biomarker of advanced fibrosis in NASH. Addition of biomarkers such as IL-6 and VCAM-1 to panels may yield increased sensitivity and specificity for staging of NASH. Nonalcoholic steatohepatitis (NASH) is a chronic metabolic liver disorder which can progress to hepatic fibrosis, cirrhosis, end-stage liver disease, and hepatocellular carcinoma. Recent estimates indicate that the prevalence of NASH in the general adult population is 3–5% , making Currently, the diagnosis and prognosis of NASH, as well as assessment of treatment response, are based on liver biopsy results. A major criticism of this approach is that liver biopsies are invasive procedures, which can be painful and associated with severe complications. Although considered the reference standard, liver biopsies are afflicted by sampling error, inter-operational variability, as well as low patient acceptance rates (reviewed in ). While Accumulation of toxic lipid species within the liver tissue associated with metabolic disturbances, insulin resistance, and inflammation can lead to hepatocellular stress, injury and cell death resulting in hepatic stellate cell (HSC) activation and upregulation of fibrogenesis. Although fibrosis is not a component of the NASH diagnosis , 8, fibrTo qualify use of biomarkers in a pharmacological clinical trial and regulatory submission, the Food and Drug Administration’s (FDA) Center for Drug Evaluation and Research (CDER) developed the Biomarker Qualification Program to establish standards for validating the analytical measurement and clinical utility of a biomarker for a specific context of use (COU) . ValidatAlthough several of these cytokines have been evaluated in previous biomarker studies of NASH , 19, no The test population were subjects enrolled in an on-going tissue and serum repository at Brooke Army Medical Center, TX. The study protocol was approved by the Brooke Army Medical Center ethics review board and conducted according to the principles of the Declaration of Helsinki. All subjects gave their written informed consent to participate in the study. All subjects underwent a liver biopsy due to the clinical suspicion of NASH. NAFLD was defined by consumption of less than 20 and 30 grams of alcohol every day for women and men, respectively and a liver biopsy showing fatty liver disease . The preA random series sample of NAFLD/NASH patient serum from the Brooke Army Medical Center repository was used in the current study. The serum levels of IL-6 and TNF-α were measured using the human V-plex Proinflammatory Panel 2 . MCP-1, MIP-1β, and eotaxin were measured using the human V-plex Chemokine Panel 1 (MSD). The serum levels of VCAM-1 and CRP were measured using the human V-plex Vascular Injury Panel 2 (MSD). All assays were modified appropriately to meet the FDA bioanalytical guidance and industry best practices. For all assays, three quality control samples were prepared in-house by spiking recombinant protein in an appropriate surrogate matrix to span the entire standard curve range. These quality controls (QCs) were utilized in addition to kit QCs to verify assay performance. The cytokine concentrations were determined from the standard curve with at least six points using a 4-parameter logistic fit curve to transform the mean light intensities into concentrations using MesoScale Discovery Workbench 4.0 .Assays were validated for accuracy and precision before sample analysis. The dynamic range for each analyte was determined by the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) as the lowest and highest quantifiable standard, respectively, within 25% of the nominal value. Assay accuracy and precision were established by quantifying 6 QCs along the standard curve range including an endogenous QC in human serum QCs at the LLOQ and ULOQ in 2–3 independent batches run on different days. A calculated concentration <±20% of the nominal value for the QCs and <±25% for the LLOQ and ULOQ QCs was set for acceptable accuracy and a coefficient of variation (%CV) <±20% the QCs and <±25% for the LLOQ and ULOQ QCs was set for acceptable precision. Analytical assay validation results are presented as Bland-Altman plots, with the average cytokine concentration plotted against the percent difference between the nominal and measured values.Results are expressed as mean±SD. Statistically significant differences were evaluated by a one-way ANOVA followed by Tukey’s post-hoc test or Chi-squared analysis where appropriate. Associations among variables were determined using the Pearson coefficient (R). Biomarker performance to distinguish differences among cohorts was analyzed by the area-under-the ROC (AUROC) with 95% confidence intervals (CI), compared to biopsy results. Statistical significance was set at p<0.05. Graphs and statistical analyses were performed using GraphPad Prism 7 .For a biomarker assay to be used for diagnostic or drug development purposes, at minimum, precision and accuracy of the assay should be verified , 22. ThePrecision and accuracy of each assay met FDA bioanalytical guidelines: accuracy  was determined to be between 80% and 120% of the nominal values for the QCs for each assay and precision (% Bias) was determined to be <20% for each assay . In addiFifty-two NAFLD/NASH patients were categorized according to fibrosis severity: none, mild or advanced . The meaChronic inflammatory biomarkers such as IL-6, CRP and TNFα were measured in NAFL/NASH patients with varying degree of fibrosis. IL-6 was increased by 1.4-fold (p<0.01) in advanced fibrosis patients compared to patients with no fibrosis, and increased by 1.1-fold (p<0.01) compared to mild fibrosis patients . IL-6 alBiomarkers of macrophage recruitment such as MCP-1, MIP-1β, and eotaxin also demonstrated little change amongst the groups of fibrosis severity . On the In the present study, a suite of pro-inflammatory cytokine and chemokine assays were analytically validated as well as evaluated in a clinical setting to demonstrate use as a biomarker for fibrosis severity in NASH patients. Analytical validation examines the performance of a test or assay. The level of validation depends on the assay COU and risk associated with the biomarker. All soluble biomarker assays in the present study were developed according to FDA guidelines  to be uset al. found a polymorphism in the IL-6 gene (-174G/C) that was more prevalent in NASH versus NAFLD patients and associated with insulin resistance [IL-6 is expressed in the liver, upregulated with NASH and plasma IL-6 concentrations positively correlate with fibrosis stage in NASH . Similarsistance , potentisistance . More resistance . Taken aet al. first demonstrated elevated VCAM-1 levels associated with hepatic fibrosis (≥F2) in a cohort of severely obese males undergoing bariatric surgery. While this patient population represents a portion of NASH subjects, the overall estimate of obesity prevalence among NASH patients is ~80% [VCAM-1 concentrations are elevated in the presence of liver diseases such as chronic hepatitis , 29 and  is ~80% . Further is ~80% , and we et al. performed a robust clinical examination of 261 biomolecules in 132 NASH patients. Diagnostic biomarkers of NASH fibrosis were determined based on data mining in a “factor module” scheme, where multiple mutually correlated results were considered as a single dataset. Within the factor module, VCAM-1 stood out as a biomarker of interest for NASH fibrosis and formed the basis of the FM-Fibro Index. In this [et al. found that FM-Fibro index had lower, although sufficient accuracy for predicting NASH-related fibrosis (AUROC ~0.70), yet excellent positive predictive value. Discrepancies between these studies included the number of patient with advanced fibrosis, as well as the number of pathologists and hepatologist reviewing the biopsy results [per se. Nevertheless, in an adult NAFLD/NASH population, VCAM-1 demonstrates robust performance as a fibrosis biomarker.Nonetheless, the trend of increasing VCAM-1 with fibrosis severity is consistent between both studies. Indeed, similar to our findings, VCAM-1 displayed an AUROC of 0.80 for discriminating fibrosis stage 2 or greater and outperformed FIB4 as an indicator of advanced fibrosis in NASH . VCAM-1  In this  and a fo In this , the FM- results . Moreove results . Althoug results . TherefoIn contrast to the IL-6 and VCAM-1 results, CRP, TNFα, MCP-1, MIP-1β, and eotaxin levels did not change with fibrosis severity. CRP is a generic inflammatory biomarker and is increased with obesity and steatosis, but not NASH severity , 40. Suret al. demonstrated that patients with NAFLD have a low-grade systemic inflammation and presented with higher serum levels of MCP-1 compared to controls [et al. also found no significant change in MCP-1 concentrations with varying degrees of liver fibrosis in a systems-biology based approach to NASH biomarker identification [Interestingly, MCP-1 gene expression is upregulated in human liver tissue sampled from NAFL patients . Haukelacontrols . In addicontrols . MCP-1 bcontrols . Howeverfication . To our fication .Incorporating inflammation and fibrosis specific factors into biomarker panels can increase performance and sensitivity. Clinical predictors of advanced fibrosis in NASH patients using innovative biomarker combinations such as FIB-C3 ; FIBROSpect test , tissue inhibitor of metalloproteinase-1 (TIMP-1)); and HA+CK18+TIMP-1 yielded AUROC of 0.86, 0.87, 0.90 respectively (reviewed in ). IntereThere are a number of study limitations that must be addressed. Due to the small sample size, patients were categorized into three groups , rather than evaluate each fibrosis stage separately. This approach was taken since only two patients were identified as F4 cirrhosis. Another limitation is that we only examined the biomarkers in a “test cohort”, and did not repeat the analysis in a separate “validation cohort”. Finally, due to the prospective nature of the study, we cannot evaluate a causal relationship between IL-6 and VCAM-1 and NASH fibrosis progression.In a single study comparison, the role of several cytokines and chemokines as biomarkers for NASH fibrosis were examined. IL-6 and VCAM-1 are a potential soluble biomarker for NASH risk stratification, able to discriminate between mild and severe stages of fibrosis. Moreover, VCAM-1 outperforms the pro-inflammatory cytokines and chemokines examined in the present study. Analytical validation is also required to satisfy regulatory guidance for biomarker use in a clinical trial. To this end, we demonstrated good accuracy and assay robustness for VCAM-1 as well as clinical value.


The interface circuit is mainly composed of an analog front-end current feedback instrumentation amplifier (CFIA) with chopper structure and a fully differential 4th-order Sigma-Delta (ΣΔ) analog to digital converter (ADC). The low-frequency 1/f noise of the TMR magnetic sensor are reduced by the input-stage and system-stage chopper. The dynamic element matching (DEM) is applied to average out the mismatch between the input and feedback transconductor so as to improve the gain accuracy and gain drift. The digital output is achieved by a switched-capacitor ΣΔ ADC. The interface circuit is implemented by a 0.35 μm CMOS technology. The performance test of the TMR magnetic sensor system shows that: at a 5 V operating voltage, the sensor can achieve a power consumption of 120 mW, a full scale of ±1 Guass, a bias error of 0.01% full scale (FS), a nonlinearity of x-axis 0.13% FS, y-axis 0.11% FS, z-axis 0.15% FS and a noise density of x-axis 250 pT/Hz1/2 (at 1 Hz), y-axis 240 pT/Hz1/2 (at 1 Hz), z-axis 250 pT/Hz1/2 (at 1 Hz), respectively. This work has a less power consumption, a smaller size, and higher resolution than other miniaturized magnetometers by comparison.Micro-electromechanical system (MEMS) magnetic sensors are widely used in the nanosatellites field. We proposed a novel high-precision miniaturized three-axis digital tunneling magnetic resistance-type (TMR) sensor. The design of the three-axis digital magnetic sensor includes a low-noise sensitive element and high-performance interface circuit. The TMR sensor element can achieve a background noise of 150 pT/Hz The earth is a huge magnetic source and scatters around the weak magnetic field (about 50 μT). So, there is a specific relationship between the size, direction of the magnetic field, and geographical position. We can achieve the high-precision GPS navigation by obtaining accurate geomagnetic field information. It is strategic and tactical for concealed combat equipment  . The ferf noise of TMR sensor and digital interface Application Specific Integrated Circuit (ASIC) for TMR sensors has not been reported. The study of a digital magnetometer based on TMR effect is significant. In the nanosatellites’ space application field, magnetic sensors are widely used for attitude control. Because of miniaturization and low power consumption the magnetic resistance-type sensor is selected in most nanosatellites [1/2 and a sensitivity of 1 mV/V/Guass [1/2. The power consumption is increased to a total number of 500 mW.Miniaturization high-performance magnetometers based on AMR (anisotropic magnetic resistance), GMR (giant magnetic resistance), and TMR (tunneling magnetic resistance) are widely used in military and civilian field. It is difficult to improve the change rate of magneto-resistance based on the principle of anisotropic scattering. TMR based on tunneling current has a lager change rate of magneto-resistance compared with GMR and AMR. The TMR magnetometer has a higher sensitivity and a wider linear range as the third generation magneto-resistance sensor, but it has a lager noise at low-frequency than GMR and AMR because of tunneling effect. At present, the research on TMR sensors still stays on the study of sensitive surface materials. The research on reducing 1/tellites . Magnetitellites ,5,6. For/V/Guass . In 2009x-axis, y-axis, z-axis. The change voltage signal is amplified by the front-stage amplifier and converted to the digital signal by a 16-bit ADC. The sampling rate of input data, output format, average reading, and zero-bias offset can be set. The measure range is up to ±2 Gauss; the resolution is 6.7 nT; the sampling speed is 10–157 sampling points/s the three-axis digital output is BCD or binary code; the baud rate can be chosen 9600 or 19,200; the serial port can be chosen standard RS485 or RS232 for single-point reading; the volume is 70 × 37 × 24 mm3; and, the power consumption is 400 mW. It has a higher resolution and a better linearity error of 0.1–0.5% FS, but it has a big volume because of detection circuit and digital processing circuit [f noise problem should be solved. The 1/f noise that is caused by a magnetic mechanism is always an important factor in affecting low-frequency magnetic detection capability of TMR sensors. According to the recently domestic and foreign research reports, high-frequency modulation method based on magnetic signal is the majority method to reduce 1/f noise in the miniaturized magnetoresistive sensor. In most of the micro/nano-satellite satellites miniaturization magnetic sensors are equipped with discrete devices, which have large volume and high power consumption. According to previous studies, the researchers used MgO materials as the barrier layer to improve the sensitivity of the TMR sensor, but the low-frequency noise cannot be reduced. The periodic modulation of the magnetic elements in the sensor at the chopping frequency have been proposed and tested for AMR and GMR. Here, we investigate the application of chopping techniques for TMR magnetic elements. The research on high performance digital interface ASIC for TMR sensors has not been reported. The study of high-precision miniaturized three-axis digital magnetic sensor system is necessary and significant.As shown in 1/2 by the vertical modulation film. The analog front-end interface and digital ASIC chip for the tunneling magneto-resistive sensor are implemented by 0.35 μm mixed signal 5 V CMOS technology. The performance test of the TMR magnetic sensor system shows that: lower power consumption (120 mW), higher resolution (250 pT/Hz1/2 (at 1 Hz)), better linearity (lower than 0.2% FS), and smaller volume (25 × 25 × 10 mm3) than other miniaturized magneto-resistance sensor.In this paper, we propose a high-precision miniaturized three-axis digital TMR magnetic sensor for nanosatellites’ space application. The TMR sensor element can achieve a background noise of 150 pT/HzThe tunneling magneto-resistance sensor element, current feedback instrumentation amplifier with chopper technique, Sigma-Delta modulator and digital decimation filter are introduced and designed in The tunneling magneto-resistance sensor element with the multilaminar structure is from Multidimension Technologies . The TMR sensor interface circuit is fabricated by 0.35 μm CMOS process and cooperated with Shanghai Huahong Integrated Circuit .2O3, located in the upper part of the anti-ferromagnetic layer [2, the area of magnetic tunneling junctions is 50 μm2. In this paper, the thickness of free layer/barrier layer/pinning layer is 10/1/10 nm. The multilayer structure of MTJ is Ta/Ru/Ta/PtMn/CoFe/Ru/CoFeB/MgO/CoFeB/NiFe/Ru/Ta, which structure is cooperated with Multidimension Technology Company. The thin film is deposited by magnetron sputtering. The MgO material is used as the barrier layer so that TMR element is more sensitive and higher resolution [The sensitive structure part of tunneling magneto-resistive sensor mainly consists of Pinning Layer, Tunnel Barrier, and Free Layer. The pinning layer is composed of a ferromagnetic layer and an anti-ferromagnetic layer (AFM Layer). The exchange coupling between the ferromagnetic layer and the anti-ferromagnetic layer determines the direction of the magnetic moment of the ferromagnetic layer; the tunneling barrier layer is usually composed of MgO or Alic layer . As showic layer . The magsolution ,14,15,16in) can be as small as a few millivolts, the common-mode (CM) voltage VCM depending on the application can be much larger and even vary at the range of a few volts during the operation, as shown in f noise, the chopper technique is applied in the circuit. To cope with varied CM voltage, the CFIA should have a common-mode rejection ratio (CMRR) greater than 120 dB. Furthermore, this CFIA is critical since it determines the overall performance of the read-out IC. To sum up, the main functions of this amplifier:in);Amplify the weak differential voltage ;High common-mode rejection ratio (>120 dB); and,High input impedance for TMR sensor.The output signal of TMR sensors is at a low-frequency (about 1 Hz) and a millivolt range. Therefore, they need amplifiers to boost such a signal to be compatible with the input ranges of Sigma-Delta Analog-to-Digital Converters. Although the differential output voltage signal of the TMR sensors  of CFIA, the target resolution of the Sigma-Delta ADC is more than 18 bits and the background noise is less than −140 dB. The power spectrum density of each part is shown in f noise caused by magnetic mechanism is always an important factor in affecting low-frequency magnetic detection capability of TMR sensor. Here, we used the high frequency modulation method based on magnetic signal to reduce the low frequency 1/f noise of TMR sensor element. The flux modulation structure is as shown in 1/2 at a modulation frequency of 5 kHz.The 1/f noise of TMR sensors is the main noise, we use the vertical modulation film structure to reduce 1/f noise of the TMR element. But, in the circuit the 1/f noise of CFIA is large at low-frequency. This chopper method is effective. The relationship between the cutoff frequency of the chopper constituted of the analog switch and the input capacitance:Because the low-frequency 1/chopf) noise is modulated to low frequency, the equivalent input voltage noise density can be shown:After the voltage noise of the analog switch is modulated by chopper-stabilized. The high frequency  is used for averaging out the mismatch between the input and feedback transconductors. The input-stage chopper frequency is 30 kHz, the resulting ripple is suppressed by a continuous-time ripple reduction loop (RRL), as shown in 1/2, as shown in Equations (2) and (3) shows that the relationship between the voltage noise density of analog switches, the chopper stabilized amplifier’s input offset voltage and the size of analog switches. At the same time, the minimum area of the analog switch is limited. In terms of tunneling magneto-resistive sensors’ weak signal, the 1/ace ASIC ,32,33. Iace ASIC ,35. The f noise is usually about 10 kHz. To achieve a corner frequency of 1 mHz, the chopper technology of input-stage amplifier is applied. Meanwhile, the corner frequency of the second-stage equivalent to the first-stage should also be less than 1 mHz. Therefore, the input-stage DC gain A01 is required:The corner frequency of operational amplifier 1/n and Vp) technology to improve the gain, and the circuit structure of Vn and Vp is shown in At the same time, the larger DC gain can effectively suppress the noise and the nonlinearity of the post-stage. The design of input-stage transconductance Gm1 and the current feedback transconductance Gm2 is particularly important, which determines the overall performance of the CFIA. To get higher gain accuracy, the circuit and layout are designed to satisfy the size and symmetry. The circuit structure of the input-stage chopper amplifier is shown in In order to maintain the SNR of CFIA, the noise performance of the Sigma-Delta modulator at an over sampling ratio (OSR) of 128 is as shown in 1/2. The sampling frequency is 1 MHz and the Sigma-Delta modulator bandwidth is 3.9 kHz. The input signal with a frequency of 30 Hz and an amplitude of −13 dB full scale is provided by the analog signal source. The output bit flow  of the designed Sigma-Delta modulator is collected by the Agilent logic analyzer 16804A  for FFT transformation, and the output spectrum of Sigma-Delta modulator is as shown in We verify the function of the modulator before testing the performance. The digital bit stream output is collected from the Sigma-Delta modulator by the oscilloscope Agilent MSO9104A . The transient response of the modulator is as shown in L + 1 [R = 32, differential delay factor M = 1, L = 5) cascaded band-pass amplitude response, we can be get that at the normalized frequency w = 0.005π, the amplitude can achieve −0.457 dB and −0.001 dB before and after compensation, respectively. The CIC compensation filter can flat the pass-band amplitude and make the transition zone become narrower. In order to further suppress high frequency noise, the FIR filter should have a narrow transition band. The FIR half-band filter has a sampling frequency of 15.6 kHz; a pass-band cut-off frequency of 3 kHz; a stop-band cut-off frequency of 3.5 kHz, and a pass-band attenuation of 0.001 dB. In view of the design of Sigma-Delta ADC post-stage digital filter, the three stages cascade structure  low-pass filter) is selected. After oversampling and noise shaping, the digital signal should be sampling down and filtered. The sampling frequency is reduced to Nyquist frequency. The three stages cascade circuit structure is applied in the filter. The first-stage uses a cascade integral comb (CIC) filter for primary filtering and reducing to a factor of 32 desample frequency; the second-stage uses a CIC compensation filter for ripple compensation and reducing to a factor of 2 desample frequency; the third-stage uses a FIR Half-band filter for high-frequency noise filtering and reducing to a factor of 2 desample frequency. For maximum linearity, it employs a single-bit feedback DAC. The digital signal process chip STM32F405DSC  used for the temperature and linearity compensation. The data transmission to upper computer is achieved by MAX232ESE . The interface communication mode is SPI and RS232. The 4th-order topology structure is applied in the front-stage Sigma-Delta modulator, the first-stage CIC filter cascading number should be L + 1 ,37,38. T3. The 5 V power supply is supported by the Agilent E3631 and the power dissipation of the TMR sensor system is 120 mW. The magnetic field is adjustable by the constant-current source Kenwood PW36-1.5ADP . The three-axis fluxgate magnetometer FVM-400  is useful for measuring the value of magnetic field. The whole test is in a three-layer shield environmental, as shown in The test platform of the three-axis digital TMR magnetic sensor system is shown in 5 nT). The test results of the three-axis linearity are x-axis 0.13% FS, y-axis 0.11% FS, z-axis 0.15% FS, respectively. The PSD of the TMR magnetometer is processed by a standard Matlab program. The TMR magnetometer system achieves a noise density of x-axis 250 pT/Hz1/2 (at 1 Hz), y-axis 240 pT/Hz1/2 (at 1 Hz), z-axis 240 pT/Hz1/2 (at 1 Hz) respectively, which is limited by the low-frequency noise of the TMR sensor element.The test of the three-axis linearity is as shown in 1/2 (at 1 Hz). The interface circuit is implemented by a standard 0.35 μm 5 V CMOS process. The measurement results show that the TMR sensor system can achieve a better performance as below: at 5 V operating voltage, the sensor system can achieve a power consumption of 120 mW, a full scale of ±1 Guass, a bias error of 0.01% FS, a nonlinearity of x-axis 0.13% FS, y-axis 0.11% FS, z-axis 0.15% FS, and a noise density of x-axis 250 pT/Hz1/2 (at 1 Hz), y-axis 240 pT/Hz1/2 (at 1 Hz), z-axis 250 pT/Hz1/2 (at 1 Hz), respectively. The performance of the TMR sensor element and interface circuit are both beyond Honeywell HMR2300. This magnetic sensor system satisfies the performance requirements in nanosatellites’ space application.We proposed a high-precision miniaturized three-axis digital tunneling magnetic resistance-type sensor for nanosatellites’ space application. The vacuum-packaged sensitive element by flux chopper can achieve a low background noise of less than 150 pT/HzAs shown in


Introduction: Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum β-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against β-lactams, underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of β-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA).Materials and Methods: Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical Enterobacterales isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC β-lactamase inhibitors and cephalosporins, which indicates β-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 h at 35 ± 1°C. Afterward, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without β-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed.Results: Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. These results, obtained after 4 h of incubation, were comparable with those of BMD and showed a higher accuracy than CDT.Discussion: We propose a novel phenotypic method for detection of ESBL and AmpC β-lactamases in Enterobacterales that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing. Klebsiella pneumoniae strain  and next generation sequencing (NGS), have gained relevance in clinical laboratories in the last decades . They alPhenotypic approaches to detect ESBL and/or AmpC are based on the detection of synergy between β-lactam agents and specific substances that inhibit each enzyme type. In the broth microdilution (BMD), synergy is indicated by a substantial decrease of the minimum inhibitory concentration (MIC) of the β-lactam . FollowiMethods based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can also be applied to identify β-lactam resistance. One of these approaches relies on the detection of products resulting from the hydrolysis of β-lactam antibiotics by β-lactamases . NonetheEnterobacterales adapting the principle of the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) previously described  were consecutively collected. In total, 50 strains were tested, 25 belonging to each of the two groups defined by EUCAST depending on the mechanism of resistance most likely involved ; group 2, AmpC . As a reference, three resistant control strains recommended by EUCAST for the detection of ESBL and AmpC production were tested , as well as one negative control strain (E. coli ATCC 25922) (involved : group 1C 25922) . BacteriA screening panel was developed on a 96-spot format following the layout depicted on Figure Stock solutions were prepared following the guidelines of the Clinical and Laboratory Standards Institute (CLSI)  by mixin5 CFU/ml) were pipetted onto an MBT Biotarget 96 . Sterility and growth controls were spotted on a second target. Targets were incubated for 3 or 4 h at 35 ± 1°C using a plastic transport box  serving as a humidity chamber in order to prevent the microdroplets from evaporating. After incubation, the remaining liquid was removed from the spots using filter paper, . After overlaying the spots with 1 μl of α-cyano-4-hydroxycinnamic acid matrix including internal standard , MALDI-TOF MS spectra were acquired on a microflex smart instrument . The method was performed in triplicate.DOT-MGA was performed as previously described . BrieflyThe spectra obtained from the MALDI-TOF MS acquisitions were processed using the MALDI Biotyper Software 3.1 , resulting in an identification score for each spot analyzed. A score ≥2.0 was considered as an indicator of bacterial growth detection. For each antimicrobial substance or substance combination, the minimum antibiotic concentration showing no bacterial growth, equivalent to a score <2.0, was defined as the MIC. In case all spots of one dilution series displayed scores <2.0, the MIC was defined as ≤0.25. MICs were calculated in triplicate, as the assay was simultaneously performed on three identical targets for each strain. The median MIC  was used for further analysis.The MICs of cephalosporins determined in absence and presence of ESBL and/or AmpC inhibitor were compared in order to detect synergy, which was considered to be present when a ≥eightfold reduction of the MICs was observed after addition of clavulanic acid and/or cloxacillin. The results obtained for each cephalosporin were interpreted using a computer-based algorithm following the EUCAST criteria , obtainiA genotypic characterization of all tested strains was performed with the PCR microarray Check-MDR CT103 XL . For an overview of the genes detected see Supplementary Table The screening panel was reproduced on microtiter plates by performing BMD according to the guidelines of The following combination disk tests  were carried out following the procedure recommended by the manufacturer: D63C , D67C  and D69C . Briefly, bacterial suspensions with a density equivalent to 0.5 McFarland standard was spread on Mueller-Hinton agar plates . Disks were then placed onto the inoculated medium, leaving enough space for inhibition zones to be seen correctly. Plates were then incubated at 35–37°C for 18 h, after which the diameter of the zones of inhibition was measured and recorded according to the instructions of use. The results of the three tests were interpreted as shown in Supplementary Table According to EUCAST’s guidelines , genotypIn preliminary experiments, DOT-MGA was performed on four well-characterized reference strains recommended by EUCAST for detection of ESBL and AmpC production . After 3Enterobacterales isolates displaying resistance against third generation cephalosporins were tested in order to further evaluate the detection performance of DOT-MGA. The additional genotypic testing by PCR allowed the identification of an OXA-48-producing K. pneumoniae strain , BMD, CDT and PCR in the following number of isolates, respectively; ESBL, 17,19,18,18; AmpC, 19,22,15,18; ESBL+AmpC, 1,1,0,1; none, 13,8,17,13. A detailed overview of the results yielded by each method can be found in Supplementary Table The following positive and negative percent agreement values (PPA/NPA) were obtained for the detection of each resistance mechanism by DOT-MGA: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. Percent agreement values of BMD and CDT were also calculated and are presented in Table The proposed approach offers an “all-in-one” screening method following the recommendations of EUCAST. It allows testing for different resistance mechanisms in a single step and displays higher PPA and NPA values than CDT, a well-established phenotypic test commonly used in routine laboratories. Furthermore, it yielded results comparable to those of BMD, while requiring an incubation period 14 h shorter. Since this method is not based on the detection of hydrolytic β-lactam products, it bypasses the challenges faced by other MALDI-TOF MS-based approaches that rely on this principle.DOT-MGA was able to identify the production of ESBL in 17 of 18 isolates identified as positive by PCR. The remaining isolate displayed an indeterminate DOT-MGA result. This seems to be related to factors inherent to the strain in question, given that no significant growth was detected after 4 h of incubation when the assay was performed. Hence, it was not possible to identify any synergistic effects and, thus, any resistance mechanisms. The production of ESBL by this strain was confirmed by BMD after 18 h of incubation. The results of CDT were inconclusive as all three kits were positive. False negative results for slow growing strains shows to be one limitation of our method, since it relies on bacterial growth as do several other rapid phenotypic approaches.E. aerogenes isolate. Despite the high accuracy of the PCR, this was employed in our study as imperfect reference standard  developing targets coated with lyophilized antibiotic substances according to the panel’s layout, requiring only the addition of bacterial suspension to the spots; (ii) programming a new function within the existing Biotyper software in order to analyze the growth scores of each spot, programming it to follow an algorithm based on fix criteria Figure  in orderCC-M, EI, and KB designed the experiments. CC-M and EI performed the experiments. CC-M, EI, KS, and MK designed and analyzed specific MALDI-TOF MS instrument settings for experiments. CC-M, EI, KS, and KB analyzed the data. CC-M and EI wrote the manuscript with input from KS, MK, and KB. All authors reviewed and edited the manuscript.EI and KB are inventors of a pending patent, which is owned by the University of Münster and licensed to Bruker. KS and MK are employees of Bruker Daltonik GmbH. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ability to recognize the molecular chirality of enantiomers is extremely important owing to their critical role in drug development and biochemistry. Convenient discrimination of enantiomers has remained a challenge due to lack of unsophisticated methods. In this work, we have reported a simple strategy for chiral recognition of thiol-containing amino acids including penicillamine (PA), and cysteine (Cys). We have successfully designed a nanoparticle-based chemiluminescence (CL) system based on the reaction between cadmium telluride quantum dots (CdTe QDs) and the enantiomers. The different interactions of CdTe QDs with PA enantiomers or Cys enantiomers led to different CL intensities, resulting in the chiral recognition of these enantiomers. The developed method showed the ability for determination of enantiomeric excess of PA and Cys. It has also obtained an enantioselective concentration range from 1.15 to 9.2 mM for PA. To demonstrate the potential application of this method, the designed platform was applied for the quantification of PA in urine and tablet samples. For the first time, we presented a novel practical application of nanoparticle-based CL system for chiral discrimination. Enantiomers of chiral molecules may express extremely different effects in terms of biochemical activities, potency, mechanism, metabolic pathways3, stereo-specific synthesis, production of pharmaceuticals2, toxicity, and metabolism1. For instance, in biological processes, enantiomers generally exhibit different pharmacological functions and toxicities on living organisms4. Human body responses differently to different enantiomers of some chiral compounds and only one enantiomer may interact desirably, while the other may induce serious dangerous effects5.Chiral recognition is considered as one of the most essential yet difficult measurements of molecular recognition. Chiral identification is a key process in fields of biochemistry, biotechnology, modern chemistry, biology, development of asymmetric catalysts4, and cystinuria5. It is also an important intermediate in many pharmaceutical syntheses. In addition, D-PA can be used as an antidote in some heavy-metal poisoning. In contrast, L-PA has been found to have toxic properties since it may contribute to several considerable adverse reactions like osteomyelitis and neuritis6. lt can also inhibit the action of pyridoxine7. Another thiol-containing amino acid, cysteine (Cys) has vital biological functions in neuronal tissues, metabolism, brain, detoxification, and protein synthesis10. Enantiomers of Cys express extremely different effects on fundamental physiological processes. As an example, L-Cys is essential for living beings and D-Cys is useful in tumor treatment11. Cys is useful for protecting normal tissues against the undesirable side effects of cancer chemotherapeutic agents and radiation treatment. Radioprotective and chemotherapeutic strategies might be developed using the D-Cys12. L-Cys aids many substantial cellular functions such as metabolism and detoxification. Low levels of L-Cys normally acts as a neuroprotective antioxidant in neuronal activity, while high levels of L-Cys may cause neurotoxic effects that may lead to following neuronal trauma like brain ischemia. In contrast, D-Cys does not lead to excitotoxic damage to the brain and protects cerebellar neurons from oxidative stress induced by hydrogen peroxide8. Thus the development of a simple method for chiral recognition of Cys and PA, and determination of their enantiomeric excess is extremely important in the fields of pharmaceutical science clinical medicine, and biochemistry.Penicillamine (PA) is a critical chiral drug for many therapeutic applications. It exists in two isomeric forms, including D-penicillamine (D-PA) and L-penicillamine (L-PA). The important pharmaceutical form is D-PA which is used in the treatment of Wilson’s disease, rheumatoid arthritis, and hepatitis. D-PA is also applied for the prevention of infants’ retina disease in preterm infants13, biamperometry14, fluorometry18, high-performance liquid chromatography20, and spectrophotometry24 have been proposed for the identification of D-PA. However, these methods cannot make an effective discrimination between D- and L- enantiomers. On the other hand, various analytical techniques, including high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), capillary electrophoresis (CE), gas chromatography, mass spectrometry36, circular dichroism (CD)37, fluorometry38, nuclear magnetic resonance (NMR) protocols40 and using chiral light fields44 have been reported to distinguish the chirality of PA, Cys, and other chiral compounds. However, most of these techniques have several drawbacks such as laborious setup process, expensive chiral columns, complicated sample pretreatment, complex operation process and high-cost chiral stationary phases or chiral selectors. Therefore, the design of inexpensive and simple methods for chiral recognition of enantiomers is still a great challenge.Different analysis methods including CL48. Chemical sensing based on nanoparticles has attracted research interests due to their unique size-dependent properties. The catalytic effect of NPs also leads to excellent CL signal amplification, leading to their efficient use in chemiluminescence (CL) sensors. Because of simple, inexpensive, and low background signal features, NP enhanced-CL systems can be used as efficient sensors52. There are few reports on NP-based recognition of chiral compounds58 but to the best of our knowledge, no study has so far been published on NP-based CL sensor for chiral discriminations.Intrinsic chirality of nanoparticles(NPs) and the relationship between the chirality of nanoparticles and chirality of molecules on their surface are interesting topics that have been extensively studied in the past years2O2 CL system to generate strong CL radiation59. In the present work, a new CL method based on TGA-CdTe QDs has been proposed for the discrimination of PA and Cys enantiomers. It was found that different affinity of L- and D- enantiomers of PA and Cys towards CdTe QDs leads to different CL intensities, resulting in the chiral recognition of these thiol-containing amino acids.As we have reported previously, thioglycolic acid (TGA) capped cadmium telluride QDs (TGA-CdTe QDs) can act as an enhancer for the luminol–H2O2 system in the presence of PA enantiomers were investigated. PA enantiomers were added to QDs solution. After the interaction of PA enantiomers with QDs, the mixture solution was injected into the ultrapure water as a carrier stream through a loop-valve injector, mixed with luminol and H2O2 solutions through three-way pieces and the CL signals were recorded  that is higher than that of L-PA/ CdTe QDs . In addition, our surface-charge density analysis revealed that zeta potentials of the CdTe QDs, L-PA/ CdTe QDs, and D-PA/CdTe QDs were −2.46, −7.96, and −18.1 mV, respectively that shows more interaction of D-PA with CdTe QDs compared with L-PA. Therefore we reasoned that more affinity of D-PA to QDs causes that a higher number of D-PA molecules attach to QDs and less H2O2 scavenging occurs, leading higher CL intensity compared with L-PA.In the present work, we have demonstrated a novel application of QDs catalyzed CL system for chiral discrimination and determination of enantiomeric excess of D/L- PA and Cys based on the difference in the CL intensities. The circular dichroism (CD) measurement confirmed that L- and D-PA and Cys exhibited mirror-image profiles see Fig. . CL respsee Fig. . Interes65. On the other hand, it has been widely studied that NPs can enhance luminol–H2O2 CL intensity by catalyzing the decomposition of H2O266. CdTe QDs have been utilized to significantly catalyze the CL systems such as luminol–H2O2 CL system67. The catalytic effect of NPs is related to their sizes and it increases with the available surface area of the catalyst50. The surface areas of the NPs decrease with increasing particle size that caused by aggregation and therefore decrease their catalytic efficiencies. Based on the enhancement effect of QDs in luminol-H2O2 CL system and considering the more aggregation of QDs by D-Cys isomer, it is expected that the less CL intensities be observed as a result of the less exposed surface area of QDs for catalytic effect. The clear difference in the results of the two enantiomers suggests that our simple CdTe QDs-based CL assay is capable of enantiorecognition of PA and Cys. Zhou et al.64 reported that through the strong interaction between thiol and the Cd atom, cysteine and penicillamine could attach to the CdTe QDs by building Cd-S bonds. On the basis of their empirical observations and calculation results based on the CdTe atomic model, they concluded that D-isomers (Cys or PA) have a stronger interaction with CdTe compared with L-isomers (Cys or PA). We also observed these different affinity interactions via UV-Vis and IR spectrometries  for chiral discrimination of L-PA and D-PA was obtained when the QDs concentration was 680 nM (77%) which was chosen as optimum concentration. To obtain the maximum ΔI, the effect of NaOH concentration was also investigated over the range of 0.5 to 2.8 mM. As Fig. The optimal conditions for luminol–HIn order to obtain the best CL intensity difference in presence of L-Cys and D-Cys, the incubation time was investigated. The experimental results showed that the highest ΔI for chiral discrimination of L-Cys and D-Cys was obtained when incubation time was 1 h see Fig. .2O2 scavenging, which conducted to lower CL intensity. Correspondingly, a linear relationship from 0.5 mM to 4.6 mM, and 2.3 mM to 4.6 mM was obtained for L-PA and D-PA, respectively  of PA and Cys were prepared and their effect on the CL of CdTe QDs-CL system was studied. The relationship between the enantiomeric excess of targets and CL was obtained by keeping the total concentration of two isomers the same (i.e. 2.3 mM) for this assay system. As shown in Fig. The enantiomeric excess (ee) means the absolute difference between the mole fractions of each enantiomer. The enantiomeric excess determination is necessary for many fields, such as asymmetric catalyst discovery and chiral drug production2O2 CL system towards other amino acids at the same concentration, including arginine, tryptophan, tyrosine, proline, and histidine. As shown in Figs 2O2 adding D-enantiomer was almost the same as L-enantiomer. The results indicated that proposed method has high selectivity over other amino acids enantiomers under our experimental condition. Since cysteine and penicillamine could attach to the CdTe QDs by building Cd-S bonds and differences in these interactions led to different CL intensities, the proposed method has high selectivity over other amino acids enantiomers that are lack of thiol groups.Selectivity of the method was evaluated by investigation of the response of the QDs-luminol-HIn order to assess the application of the proposed method for the detection of PA in real samples, the designed approach was applied to determine the concentration of D-PA in tablet and urine samples spiked by D-PA or L-PA. The results in Table In this work, a simple and novel strategy was utilized for chiral recognition of PA and Cys using QDs enhanced-CL system. This effective enantioselective sensing method was based on an obvious difference in the CL intensities in presence of L and D-enantiomers. The proposed chemiluminometric method demonstrated excellent performance to determine the enantiomeric excess (ee) of PA and Cys in the whole range of ee values. Moreover, the proposed method exhibited good linearity for PA quantification. Practical detection of PA in pharmaceutical tablet and urine sample was also successfully examined. In comparison with conventional analysis for chiral discrimination such as HPLC, GC, CD, and NMR, this technique has many advantages because of its simple instrumentation, low cost, and high selectivity. We believe that NP enhanced-CL systems can find important applications, including their use as chiral recognition sensors.2. 2H2O) were obtained from Sigma. Sodium borohydride , 5-amino-1,2,3,4-tetrahydro-1,4-phthalazinedione , hydrogen peroxide , and sodium hydroxide (NaOH) were bought from Merck. Penicillamine (PA), cysteine (Cys), arginine (Arg), tryptophan (Trp), tyrosine (Tyr), proline (Pro), and histidine (His) enantiomers were purchased from Sigma. Milli-Q grade water (18.2 MΩ.cm at 25 °C) was used throughout the experiments.Thioglycolic acid , tellurium powder , and cadmium chloride (CdClCircular dichroism (CD) measurements were recorded on a Jasco J-810 CD spectropolarimeter. Size distributions were obtained from Dynamic Light Scattering (DLS) by using Zetasizer Viscotec 802 at ambient temperature. UV-Vis spectra were measured and recorded with a Lambda  spectrophotometer with the use of 1.0 cm cell. FTIR spectra of samples were taken using an ABB Bomem MB-100 FTIR spectrophotometer. All the spectra were obtained at room temperature.68. Typically, 0.256 g of CdCl2·5H2O and 200.0 µL TGA were added in 4.0 mL ultrapure water. Meanwhile, 3.0 M NaOH was used to adjust the pH to 9.0. In the next step, 0.065 g of Te powder and 0.183 g of NaBH4 were added into 75.0 mL ultrapure water under argon flow at 50 °C in a three neck round bottom flask. The solution was refluxed with vigorous stirring until the solution turned purple. Subsequently, the temperature of the solution was increased to 150 °C and the prepared Cd solution was added to the purple solution. After 2 h of reaction, TGA-CdTe QDs solution was obtained. Finally, the resulting solution was stored in the refrigerator (4 °C). The as-prepared CdTe QDs were characterized by atomic force microscopy (AFM). The size of CdTe QDs was determined by AFM  and the CL signal was then monitored. The same procedure was done for L-Cys and D-Cys except that it was incubated for 1 h at room temperature. The process of enantiomeric excess detection was the same as that recognition of D- and L-enantiomers, upon addition of 300 μL Cys or PA with the different enantiomeric excess solution to 1 mL of QDs.D-PA or L-PA was dissolved in water containing 150 μL of 0.5 M of NaOH to a total 10 mL volumetric flask to obtain different concentrations of PA. 466 µL of the synthesized TGA-CdTe QDs were diluted with DI water to a final volume of 25 mL. In a 2 mL eppendorf tube, 1 mL of diluted QDs solution and 300 μL D-PA or L-PA solution at different concentrations were added, and then the mixture was incubated for 4 h at room temperature. For flow-injection chemiluminometric chiral discrimination of D-PA and L-PA, a laboratory-built flow injection CL system was used. As shown in Fig. The practical application of the proposed method was evaluated by using pharmaceutical tablets and urine samples. A fresh urine specimen was prepared from a healthy volunteer and a fifty-fold sample dilution was made. To assess the recovery and precision D-PA and L-PA were spiked into the urine sample under defined optimal conditions prior to CL measurement. A pharmaceutical tablet was dissolved in 50 mL pure water and was filtered to remove large insoluble particles. The clear solution was then subjected to the same CL assay under optimized conditions. This study was carried out in accordance with relevant guidelines and regulations and has been approved by the ethical committee of the Sharif University of Technology. Informed consent was obtained from all subjects.Supporting Information


Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder affecting 7–10 million individuals. The pathologic hallmark of PD is nigrostriatal dopaminergic neuron loss, leading to several motor and nonmotor disturbances, such as akinesia, gait disturbance, depression, and anxiety. Recent animal studies have demonstrated that physical exercise improves behavioral and neuropathological deficits in PD. However, the exact underlying mechanism underlying this effect remains unclear. In this study, we investigated whether long-term exercise has neuroprotective effects on dopaminergic nigrostriatal neurons and whether it further alleviates impairment of the gait pattern, locomotor activity, akinesia, and anxiety-like behavior in PD rats.  A hemiparkinsonian rat model, generated by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, was applied to evaluate neuroprotective effects and motor behaviors. Comprehensive spatiotemporal gait analysis, open-field locomotor activity, akinesia, apomorphine-induced rotational analysis, and dopaminergic neuron degeneration level were assessed every week and up to 8 weeks after daily voluntary running wheel exercise.  Compared with the sham-treated group, we found that 10 weeks of voluntary exercise  significantly reduced 6-OHDA-induced motor deficits in the gait pattern, akinesia, and rotational behavior in the exercise group. Immunohistochemically, a tyrosine hydroxylase-positive neuron in the substantia nigra was significantly preserved in the exercise group.  Our results demonstrated that long-term exercise training is effective for neuroprotection and further attenuates motor declines induced by 6-OHDA in an experimental model of PD. Our data further highlighted potential therapeutic effects of long-term physical exercise relevant to clinical effects for further potential application on human PD subjects. Parkinson's disease (PD) is an idiopathic disease of the nervous system characterized by progressive tremor, bradykinesia, rigidity, and postural instability. The pathologic hallmark of this movement disorder is due to the denervation of dopaminergic neurons in the nigrostriatal pathway, leading to both motor and nonmotor symptoms –3. PharmPhysical exercise is a common, practical, uncomplicated, noninvasive, and relatively safe approach for improving motor and cognitive functions and dopaminergic functioning in clinical PD patients , 8. Prev6-Hydroxydopamine (6-OHDA) is the most commonly used neurotoxin to induce PD in the rodent model , 20. WitThe present study identified the therapeutic potential of long-term effects of early voluntary exercise intervention on a PD animal model by monitoring gait, locomotor activity, akinesia, and dopaminergic nigrostriatal neurons in 6-OHDA hemiparkinsonian rats as an early step toward possible eventual clinical conditions. Therefore, strategies to investigate individual variability in lesion and injury progression may further play a pivotal role in evaluating therapeutic effects of voluntary exercise on the 6-OHDA study model.Male Sprague-Dawley rats  were housed in a temperature- (22 ± 2°C) and humidity-controlled (50%) environment and placed on a 12/12 h reversed dark-light cycle with ad libitum access to standard chow and tap water. Upon arrival, the rats were allowed to have 2 weeks of the acclimation period to the animal facility. After the acclimation period, the rats were allocated randomly and equally to two groups: experimental and control groups. All procedures were approved by the Institutional Animal Care and Use Committees of Chang Gung University  and performed following the Guide for the Care and Use of Laboratory Animals 8th edition (2011) for humane treatment at all times of the present study.μg 6-OHDA (Sigma-Aldrich) dissolved in 4 μl of 0.02% ascorbic saline into the MFB of the left hemisphere, causing a progressive retrograde and dose-dependent neuronal degeneration in the nigrostriatal pathway [μl/min by using a 10 μl Hamilton microsyringe fitted with a 26-gauge steel cannula and left for 8 min to assure the solution diffusion to prevent backflow. The wound was closed after the cannula was removed.Animals received 6-OHDA injection when the body weight increased to 350 g. Anesthesia was administered through an intraperitoneal injection of Zoletil  mixed with xylazine . The anesthetized rats were placed in a stereotaxic frame  for the injection procedures. A longitudinal incision was made along the dorsal medial surface of the head, exposing the skull from the bregma to the lambda. A 1.5 mm diameter hole was drilled using a stereotaxic high-speed drill at the following locations: −4.3 mm (AP to bregma), 1.6 mm (ML), and −8.2 mm (DV), to the MFB. Rats with MFB lesions received an injection of 8  pathway , 24. InjL) × 23.5 cm (W) × 20 cm (H)), one running wheel , and one rat only. The exercise animals had unlimited access to a running wheel in their exercise cage. The running wheel apparatus  was connected to a sensor that measured the daily rotations and converted the numbers of rotations to distance (meters). The sensor outputs were recorded and analyzed to acquire and monitor the exercise performance of rats.The running wheel for rodents was applied for exercise intervention in the present study. The exercise rat was caged in the customized exercise cage  which contained a transparent Plexiglas arena  × 6 cm (W) × 12 cm (H)) with an inclined mirror positioned at a 45° angle beneath the walkway. With a high-speed and high-resolution camera  placed parallelly to view the image of the entire walkway, sagittal gait characteristics, reflected by the mirror image, could be acquired for further pattern identification [The gait parameters that were significantly affected by 6-OHDA lesions in the nigrostriatal pathway in rats may be used to study disease progression and to test time frame effects of voluntary exercise , 19. By fication .The forelimb akinesia test, as known as the bar test, was used to evaluate the forelimb mobility in a PD rodent model , 25, 26.L) × 60 cm (W) × 80 cm (H), produced in-house), and each rat was allowed to explore the space after placing in the center of the arena for 10 min. All the activities of the animals within the arena were automatically recorded using SMART tracking software . The behavioral patterns within the arena were analyzed based on the total movement distance: distance traveled both in the center and along the perimeter of the area, time spent in the center, movement velocity, and freezing time [The open-field test was used to evaluate motor movement distance activity and anxiety –29. It wing time , 31. TheApomorphine administration induces abnormal contralateral rotations in hemiparkinsonian model rats; these were measured every week after 6-OHDA injection to estimate the severity of dopamine depletion , 32, 33.μm coronal blocks. The sections were selected from the Str and SN on a cryostat . The free-floating sections were quenched with 0.3% H2O2/PBS for 10 min and 10% milk  for 60 min to block a nonspecific antibody. Sections were then incubated with rabbit primary anti-TH  for 1 h at room temperature. Subsequently, sections were washed in PBS and incubated for 1 h with a secondary anti-rabbit antibody  containing 3% normal horse serum in PBS. After rinsing, sections were immunostained with 3,3′-diaminobenzidine  and observed for 3–5 min. Lastly, the stained sections were mounted onto slides, air-dried, dehydrated using xylol , cleared in xylene, and cover slipped in DPX mounting medium. TH-immunopositive cells in the SN pars compacta were counted manually in each section [After voluntary exercise and behavioral tests were assessed 56 days postlesion and 70 days postvoluntary exercise, animals were sacrificed for tyrosine hydroxylase (TH) staining to evaluate and estimate dopaminergic neurons in the SN. Animals were deeply anesthetized with an overdose of inhaled isoflurane in oxygen (<0.5%) , followed by an intracardial infusion of phosphate-buffered saline (PBS) and a 4% paraformaldehyde fixative solution. Brains were carefully removed, postfixed for 3 days, and cryoprotected in 30% sucrose solution at 4°C until brains sank completely. The cerebral tissues were sectioned at a thickness of 30  section  using a t-test was conducted to verify the intergroup difference, and one-way ANOVA was used to investigate intragroup differences in the time course when no significant main effect after two-way repeated measures ANOVA was observed. Data were analyzed using SPSS version 24.0 , with the significance level set at p < 0.05 for each analysis. All data were presented as mean ± standard error of the mean.For the statistical analysis of gait measurement, two-way repeated measures analysis of variance (ANOVA) was used to investigate parameter changes between both the groups (exercise vs. nonexercise) and time (week) factors. When the ANOVA showed significant effects, multiple within-subject comparisons with the Bonferroni post hoc analysis were performed to compare the effects of exercise and time courses. The independent F = 42.033, p < 0.001) and time course  but no significant effects on the group vs. time course interaction . The intergroup data for the exercise-treated group showed a significantly lower rotation number at week 2 (p = 0.047), week 3 (p = 0.043), week 4 (p = 0.03), week 6 (p = 0.014), and week 8 (p = 0.001) than that for the nonexercise control group , time course , and group vs. time interaction . The intergroup analysis indicated a significantly higher bar test score at week 4 (p < 0.001), week 6 (p < 0.001), and week 8 (p < 0.001) in the nonexercise control group than in the exercise-treated group . This indicated no significant difference between the exercise group and the nonexercise control group at the beginning of the present study. F = 15.944, p = 0.003), step length , stride length , and stance phase time , suggesting less impairment of the gait pattern in the exercise group as compared with the nonexercise group. Following post hoc t-tests between groups in each time point revealed that this difference was largely driven by exercise effects observed starting at six weeks or seven weeks of exercise on walking speed, step length, stride length, stance phase time, swing phase time, and double support time   and not on the group  and group vs. time interaction  , group , and group vs. time interaction . There are no significant differences in overall traveled distance and times in the central zone between the exercise and nonexercise groups at each time point .The present study also calculated the overall distance traveled using an open-field test to investigate the general locomotor activity between the 6-OHDA exercise-treated and nonexercise-treated rats Figures . In the = 0.755) . In the p = 0.031) (TH-positive immunoreactivity in the SN and Str of the rats with 6-OHDA lesion is demonstrated in = 0.031) .The present study investigated motor behaviors in unilateral 6-OHDA-lesioned rats in response to voluntary physical exercise to better understand the interrelation of time course exercise intervention, neuroprotective effect, and motor behaviors. We observed that long-term voluntary physical exercise training restored several PD-related motor dysfunctions and alleviated the dopaminergic neuron loss in the SN in the 6-OHDA-induced PD rat model, suggesting that exercise maintains motor function and potentially reduces dopaminergic neuron damage.Based on our detailed assessment by using the bar and rotation tests, the decrease in dopaminergic function caused by unilateral 6-OHDA MFB lesions can be investigated quantitatively and objectively , 29. TheThe present spatiotemporal gait analysis confirmed previous evidence suggesting that the PD rats with voluntary exercise in the exercise-treated group displayed a significantly higher walking speed, associated with longer step and stride lengths, than those in the nonexercise group. The walking speed of the exercise-treated group increased gradually and reached a stable plateau accompanying time course exercise intervention, which indicated that voluntary exercise improves motor function in PD animals. Moreover, these behavioral findings in the group treated with 8 weeks of voluntary exercise, accompanied by attenuation of 6-OHDA-induced degeneration of nigral dopaminergic neurons and the preservation of TH-positive fibers of the nigrostriatal pathway, strongly suggest exercise-induced neuroprotection, which has been reported in other studies , 35, 36.The present results suggest that time course voluntary exercise can have long-lasting effects on neuroprotection of dopaminergic neurons in the SN. These results are consistent with those of earlier PD animal studies, showing that physical exercise can reduce behavioral and neuropathological effects directed toward the DA neurons of the SN , 39. ThiIn the present study, rats were exercising when the behavioral tests were performed, and upregulation of DA receptors through exercise may have reduced the interhemispheric asymmetry following 6-OHDA lesion, thereby reducing apomorphine-induced rotations and increasing the walking speed in exercise-treated rats. These findings suggested that the long-lasting effects of voluntary exercise intervention may lower PD influences in the present study in the PD rodent model. Furthermore, this requires careful interpretation of findings from animal studies regarding any potential role of neurogenesis in the effects of exercise on PD patients. One of the main future challenges will be to assess and define exercise-induced effects on animal models comprehensively and to translate these results to human studies meaningfully.In summary, the results of the present study revealed the effects of 8 weeks of voluntary exercise on motor behavior and neuroprotection in the 6-OHDA rodent model. The neuroprotective effects of voluntary exercise in the present unilateral 6-OHDA rat model accompanied the motor performance in decreasing the akinesia behavior and increasing the gait performance. In association with the immunohistological results, the decrease in dopaminergic system deterioration, as observed by TH levels, indicated protective effects of voluntary exercise on dopaminergic cells after 6-OHDA injury. The present results suggest that voluntary exercise may provide long-term improvements in functional motor behavior in PD patients.


Urethane groups formed by reacting phenolic hydroxyl groups with isocyanates are known to be reversible at high temperatures. To investigate the intrinsic self-healing of polyurethane via a reversible urethane group, we synthesized vanillyl alcohol (VA)-based polyurethanes. The phenolic hydroxyl group of vanillyl alcohol allows the introduction of a reversible urethane group into the polyurethane backbone. Particularly, we investigated the effects of varying the concentration of reversible urethane groups on the self-healing of the polyurethane, and we proposed a method that improved the mobility of the molecules contributing to the self-healing process. The concentration of reversible urethane groups in the polyurethanes was controlled by varying the vanillyl alcohol content. Increasing the concentration of the reversible urethane group worsened the self-healing property by increasing hydrogen bonding and microphase separation, which consequently decreased the molecular mobility. On the other hand, after formulating a modified chain extender (m-CE), hydrogen bonding and microphase separation decreased, and the mobility  of the molecules improved. In VA40-10  heated to 140 °C, the self-healing efficiency reached 96.5% after 30 min, a 139% improvement over the control polyurethane elastomer (PU). We conclude that the self-healing and mechanical properties of polyurethanes might be tailored for applications by adjusting the vanillyl alcohol content and modifying the chain extender. Polyurethane elastomer (PU) is a versatile polymer with many industrial applications. PUs is generally synthesized from polyols, diisocyanate, and a chain extender by the prepolymer method. The physical properties of PU can be adjusted by varying the types and amounts of polyols, isocyanates, and chain extenders in the precursor mixture. PU is a segmented block copolymer composed of a soft domain (polyols) and a hard domain (isocyanate and the chain extender). The polyol in the soft domain gives the PU its elastic quality, while the hard domain (depending on its nature) confers hardness and rigidity. Thus, microphase separations and the contents of the soft and hard domains significantly affect the mechanical properties of PU. Thermal stability of PU has also been reported. Chain scission occurs in the hard segment when PU is heated above 200 °C ,2,3. PU Recently, many studies have focused on the self-healing and shape memory properties of PU. Self-healing PUs are materials capable of repairing damage via release of healing agents from embedded microcapsules or a temporary increase in mobility leading to re-flow of the material in the damaged area. Compared to other materials, there are many choices to impart self-healing properties to Pus, as the physico-chemical properties of PU can be adjusted with the types and amounts of polyols, isocyanates, and chain extenders. Many researchers are improving the durability and applicability of polymer products by enhancing their self-healing properties. Various fabrication methods for self-healing polymers have been suggested. In early research, self-healing was extrinsically enhanced by dispersing microcapsules in the polymer . When crTo overcome these problems, later researchers developed intrinsic self-healing methods. These methods can be categorized into two types. Associative self-healing methods include hydrogen bonding, host–guest interactions, or exchange reactions involving transesterification, disulfide metathesis, transalkylation, transcarbamoylation, or azomethine metathesis ,11,12,13However, applicability of the above-mentioned healable polymers with reversible urethane bonds are limited by various conditions. For instance, polymers that self-heal only at high temperatures under inert gas conditions are unsuitable for low-temperature applications. In the present study, we investigate whether the concentration of the dynamic covalent bonds influences the self-healing property of PU. Typical urethane bonds constitute aliphatic hydroxyl and isocyanate groups, which are stable and indecomposable below 200 °C. The phenolic hydroxyl group can also react with the isocyanate group, blocking the isocyanate and improving the storage stability of the prepolymer. Urethane groups constructed from phenolic hydroxyl and isocyanate can re-dissociate into phenol and isocyanate via a reversible reaction. Inspired by these facts, we chose vanillyl alcohol (VA) as the diol and mixed it with a polyol to synthesize a VA-based PU with two hydroxyl groups  that self-heals under heating. VA enables the introduction of a reversible urethane group composed of a phenolic hydroxyl with an isocyanate group into PU. The mechanical and self-healing properties of PUs with different concentrations of the dynamic chemical bonds are investigated. In addition, the self-healing properties of PU are improved by a modified chain extender (m-CE) that improves the mobility of the molecules.−1, respectively. Hydrogen-bonded and free hydroxyl groups of VA were observed at 3340 and 3160 cm−1, respectively. VA-based PUs synthesized by the pre-polymer method are illustrated in −1 in the FT-IR spectrum of the VA-based PUs disappeared after curing at 110 °C for 24 h  . VA-baseTgs) manifested as a peak in the tan delta curve. In Tgs increased with increasing VA concentration in the PU, reflecting the decreased content of the polyol constituting the soft domain and the increased content of the urethane group capable of hydrogen bonding (per unit mass). Meanwhile, the Tgs of the PUs with m-CE in flowT), at which the storage modulus of the PUs decreased after the rubbery plateau region, was lower in the VA-based PUs than in the control PU, and it increased with increasing VA content. This implied that the VA hindered the microphase separation, and dissociation of reversible urethane groups was lower than Tflow of the control PU. However, Tflow in the VA-based PU increased as the VA content increased because the hard segment concentration increased. Dynamic mechanical properties in the PUs were investigated by a dynamic mechanical analyzer (DMA). The results are displayed in Tgs) and Tflow of the VA-based PUs, again by interfering with the hydrogen bonding and hard segment packing (confirmed by FT-IR). The stress relaxations in the PUs were investigated by employing DMA, and the test results are given in At low temperatures, hydrogen bonds increased the storage modulus of the VA-based PUs over the control PU (as confirmed by FT-IR). In Tgs increased with increasing VA content. More specifically, Tgs was −45 °C in the control PU and −43, −40, −36, and −20 °C in the VA10, VA20, VA30, and VA40 samples, respectively. The hard domain melted at approximately 150 °C. However, when the hydrogen bonds were disrupted by m-CE, Tflow decreased. For instance, the Tgs and melting temperature of VA40-10 were −30 °C and 143 °C, respectively. The increase in Tgs with VA content was attributable to phase mixing and hydrogen bonding by VA . Stress–strain properties of the PUs were studied in tensile tests, and they are presented in Although VA-based PUs showed earlier stress relaxation at elevated temperatures (above 120 °C) than at low temperatures, their tensile properties at room temperature were superior to those of the control PU. Self-healing properties of the samples were evaluated on dog-bone specimens. The center of each prepared specimen was cut with a knife, and the two halves were immediately reattached. The self-healing process is imaged in Butyl glycidyl ether , diethanolamine , poly(tetramethylene ether)glycol  (PTMEG1000), and 1,4-butanediol  were obtained from Sigma–Aldrich. Vanillyl alcohol  was purchased from Tokyo Chemical Industry in Korea. 4,4’-methylene diphenyl diisocyanate  was obtained from BASF .−1 completely disappeared after the reaction. In the H1-NMR spectrum  with BGE . The mixture was stirred at room temperature for 24 h. The mixture was initially opaque but gradually became transparent through the exothermic reaction. spectrum , the peaVA-based PUs were synthesized by the prepolymer method. The prepolymer was synthesized by reacting 0.1 mol of PTMEG1000 and 0.2 mol of MDI at 70 °C for 3 h. The synthesized prepolymer was mixed with the desired amounts of MDI, BDO, m-CE, and VA. After degassing, the mixtures were poured into a glass mold to prepare PU films and cured at 110 °C for 24 h in a convection oven. PU films obtained after the cure were employed for characterizations.1-NMR spectra were recorded in chloroform-d with a Bruker AM400 spectrometer (600 MHz). Molecular weights of the VA-based PUs were determined by GPC  employing a refractive index detector . Samples were dissolved in DMF/THF (1:1 wt./wt.), and polystyrene standards were used for universal calibration. The flow rate of the GPC measurement was 1.0 mL/min. Thermal properties of the VA-based PUs were measured by DSC . The samples were initially cooled to −90 °C and maintained at this temperature for 1 min. In all measurements, the temperature was ramped up to 240 °C at 10 °C/min. A second scan was conducted under the same conditions. All tests were performed in an N2 atmosphere. Dynamic mechanical properties and relaxation times of the VA-based PUs were measured by DMA  using a film tension clamp. The films for DMA were prepared by carefully cutting the cured PU films into 5.3 mm-wide sections. Dynamic mechanical properties were measured between −90 and 240 °C inclusive at a heating rate of 5 °C/min. Relaxation tests were also performed at 110, 120, 130, 140, 145, and 150 °C for 20 min under an axial force of 0.01 N and a deformation of 1%. Tensile strengths and percentage elongations were measured by a universal testing machine  following the ASTM-D638 method. SAXS patterns  were collected at room temperature using Cu–Kα radiation (λ = 1.541 Å) at a voltage of 50 kV and a current of 1000 µA. The Bragg angle (2θ) ranged from 0° to 9°. Synthesis of the m-CH and VA-based PUs were confirmed by Fourier-transform infrared (FT-IR) spectroscopy . H1703/I1733 ratio in the FT-IR spectrum, which indicated the extent of hydrogen bonding in the PU, significantly decreased to 0.92 in VA40-10. On the other hand, the I1703/I1733 value of VA40 (without the m-CE) was 2.21, indicating strong hydrogen bonds in this sample. The interdomain distance in the SAXS profile of VA40-10 was only 14 nm, reflecting decreased microphase separation. Meanwhile, the relaxation time of the VA-based PUs at 140 °C increased from 71 to 162 s with increasing VA content. But after incorporating the m-CE, it significantly decreased, reaching 65 and 59 s in the VA40-5 and VA40-10 samples, respectively. This result was also closely related to microphase separation and hydrogen bonding in the PUs. Thus, disrupting the hydrogen bonds and microphase separation notably affected the self-healing properties of the PUs. Reduced hydrogen bonds and lower microphase separation improved the mobility of the molecules. Thus, reducing the molecular weight by dissociating the reversible urethane bonds efficiently contributes to the self-healing of PUs. To ensure efficient PU self-healing, the molecular structure design should increase both the molecular mobility and the concentration of dynamic covalent bonds.This study investigated the self-healing of VA-based PUs via reversible urethane bonds formed by the reaction of phenolic hydroxyl and isocyanate groups, which can be efficiently exploited for self-healing PUs. In particular, we investigated the influence of VA and m-CE contents on the self-healing properties by measuring the thermal and dynamic mechanical properties of the prepared samples. We confirmed that the urethane bonds can be reversibly dissociated and that the isocyanate groups regenerated above 140 °C. As the VA content of the polyols increased from 10% to 40%, the relaxation time reduced from that of the control PU. Increasing the VA content decreases the self-healing efficiency by increasing the hydrogen bonding of hard segments and decreasing the proportion of soft segments, thereby reducing the mobility of the molecules. To overcome this problem, molecular mobility was increased by incorporating m-CE into the VA-based PUs. Hydrogen bonding and hard domain packing, which improved the mechanical properties but hindered the molecular movements, was reduced by the m-CE. Consequently, the I


Previously, we found that high levels of soluble insulin receptor (sIR) in the cerebrospinal fluid (CSF) of an HIV-infected women cohort were associated with the presence and severity of HIV-associated neurocognitive disorders (HAND). In this study we investigated if CSF from this population, HIV-1 Tat, and selected cytokines induces sIR secretion from human neuronal cells. Twenty-three (23) HIV-seropositive women stratified by cognitive status and five HIV- seronegative women were evaluated. Soluble IR levels were measured in the extracellular medium of neuronal cells (SH-SY5Y) that were exposed (for 24 h) to the CSF of patients. The levels of sIR, HIV-1 Tat, and cytokine levels  were quantified in the CSF of participants by ELISA and flow cytometry. Neuronal secretion of sIR was measured after exposure (24 h) to HIV-1 Tat (0.5–250 nM), or specific cytokines. The effects of TNFα and HIV-1 Tat on sIR secretion were also evaluated in the presence of R7050 . Neurons exposed to the CSF of HIV-infected women had higher sIR levels according to the severity of neurocognitive impairment of the participant. Increased CSF sIR levels were associated with the presence and severity of HAND and were positively correlated with CSF HIV-1 Tat levels in HIV-infected women with cognitive impairment. CSF levels of IL-2, IFNγ, and TNFα were significantly increased with HAND. However, only TNFα (5 pg/mL) and HIV-1 Tat (100 nM) induced a significant increase in neuronal sIR secretion after 24 h exposure, an effect that was antagonized when each were combined with R7050. Our data suggests that TNFα and HIV-1 Tat from the CSF of HIV-infected women may regulate the secretion of sIR from neuronal cells and that the effect of HIV-1 Tat on sIR secretion may depend on TNFα receptor activation. Chronic inflammation in HIV-infected individuals has been associated with insulin resistance, cardiovascular diseases, thrombosis, muscle wasting, neurocognitive impairment, renal and liver diseases, obesity, and premature aging –10. ConsHIV-1 Tat is continuously produced in patients even in the presence of viral suppression, thus promoting the persistence of chronic inflammation in both the peripheral and the central nervous system . HIV-1 TOverproduction of pro-inflammatory cytokines has also been implicated in the pathophysiology of insulin resistance and metabolic disorders , 30. TNFIt is known that people living with HIV (PLH) may develop insulin resistance, HAND, or both, as a result of chronic HIV infection, long-term use of combined antiretroviral therapy (cART), aging, genetic predisposition, or a combination of these factors –37. Thusn = 11), asymptomatic impairment , and symptomatic impairment .This was a retrospective cross-sectional study using patient-database information and the sample repository of the Hispanic-Latino Longitudinal Cohort of Women. This study was approved by the University of Puerto Rico Medical Sciences Campus (UPR-MSC) Institutional Review Board, and all the participants signed a written informed consent. This is a unique cohort of Hispanic HIV-infected women characterized longitudinally with viral and immune profiles, neurological exams, and neuropsychological tests. Twenty-four HIV-infected women without a history of diabetes and 5 HIV-seronegative controls were evaluated, as described previously –43. Cognp > 0.05). Most HIV-infected women were using antiretroviral therapy (ART)  with protease inhibitors . There was no significant difference between groups of increasing HAND severity in either use of ART, protease inhibitor, nor CNS Penetration Effect  to confirm the absence of all sIR in the CSF of all the participants.5 cells/well) in 24-well plates supplemented with 10% FBS and 1% penicillin-streptomycin in EMEM growth medium at 37°C and 5% CO2. After allowing the cells to adhere overnight, medium was removed and replaced with sIR-depleted CSF (200 μl) from each HIV-infected patient and controls for 24 h. To determine cell viability after incubation with CSF overnight, we performed a MTT assay. Our data demonstrated that cell viability was above 95% after incubation with the CSF (data not shown). The levels of sIR in the CSF were determined by ELISA .Human neuronal cells  were grown (5 × 10n = 20) by sandwich ELISA as described previously (HIV-1 Tat levels were measured in the CSF of HIV-infected women (eviously  at Dr. AThe human TH1/TH2 Cytometric Bead Array (CBA) kit  was used to determine the levels of IL-2, IL-4, IL-6, IL-10, IFNγ, and TNFα in the CSF of HIV-infected women and HIV-seronegative controls. With the CBA, different bead populations with distinct fluorescence intensities had been coated with capturing antibodies specific for different cytokines. After incubation with 50 μL of CSF, the cytokine-captured beads were mixed with phycoerythrin-conjugated detection antibodies to form sandwich immune-complexes. Cytokine levels were performed blinded to group cognitive status and analyzed by flow cytometry.2. Cells were also exposed to different HIV-1 Tat concentrations (0.5–250 nM) for 24 h. The secreted sIR levels were analyzed by ELISA. In other experiments, secreted sIR levels were determined after incubation with TNFα (5 pg/ml) or HIV-Tat protein (100 nM) in the presence of R7050 (10−8 M).The physiological levels of cytokines in the CSF of HIV-infected women were quantified by flow cytometry, as described above. Cytokines  were added individually to the culture medium of SH-SY5Y neurons for a period of 24 h at 37°C and 5% COSoluble insulin receptor full-length  levels methods were previously determined and published , 42. BriAll the flow cytometric analyses were carried out using a FACSCalibur cytometer . CellQuest software  was used for data acquisition and multivariate analysis. Cells were gated in forward/side scatter dot plots. PE emissions from cytokines were measured in the FL2 (585 nm band-pass filter) channel. Data on scatter parameters and histograms were acquired in log mode. Ten thousand events were evaluated for each sample, and the median peak channel obtained from the histograms was used to determine the levels of CSF cytokines.p < 0.05.We compared sIR, cytokine, and HIV-1 Tat levels in the CSF of four groups: the control group and the groups of HIV-infected women stratified by cognitive impairment as having normal cognition (NC), asymptomatic impairment (AI), or symptomatic impairment (SI). Because the data were not normally distributed, these comparisons were made using non-parametric analyses. The control group consisted of 5 CSF samples. The use of 5 control samples was adequate to estimate the median and interquartile range. Non-parametric analysis was performed to compare sIR levels secreted from cultured neurons exposed to the CSF of HIV-infected women (stratified by HAND severity) and controls. Parametric one-way ANOVA analysis were used to compare neuronal sIR secretion after exposure to selected cytokines, HIV-Tat protein or combinations with and without R7050. All statistical analyses were performed with IBM SPSS Statistics (version 24) and GraphPad Prism 7. Statistical significance was considered p = 0.002). When HIV-infected women were stratified by HAND, higher levels of CSF sIR were associated with the presence (p = 0.018) and increased severity of HAND . When the CSF of HIV-infected women was stratified by HAND, higher levels of full-length sIR secreted from neuronal cells were associated with the presence (p = 0.004) and severity of HAND  by sandwich ELISA as described previously , IFN-γ (p = 0.002), and TNF-α (p = 0.009) were associated with the presence and severity of HAND (p = 0.061), a significant difference was observed between HIV-infected women with normal cognition and those with symptomatic neurocognitive impairment in a post-hoc analysis .The levels of IL-2, IL4, IL-6, IFNγ, TNFα, and IL-10 were quantified in the CSF of HIV-infected women and controls. Significantly increased levels in the CSF of inflammatory cytokines IL-2 . Among these cytokines only TNFα was able to significantly increase the levels of sIR secreted from neurons compared to those untreated  to determine the dose that would induce maximal sIR secretion by neurons. We observed a dose-dependent effect of HIV-1 Tat on sIR secretion with maximal response at 100 nM concentration , brain tissue injury, or plasma sIR that crosses the BBB . We prevAlthough the generation of intact and other sIR isoforms was observed in several cultured cell lines , their sIncreasing evidence suggests that inflammation is actively involved in the neuropathogenesis of HAND, and several cytokines have been identified as potential contributors. The importance of these cytokines, especially TNFα, has been emphasized in the propagation and maintenance of a CNS inflammatory response. We were able to quantify six cytokines in the CSF of our cohort of HIV-infected women and observed that the pro-inflammatory IL-2, IFNγ, TNFα, and the anti-inflammatory IL-10 were associated with the presence and severity of HAND. These results are consistent with other studies that show an association between cognitive function and increased levels of IFNγ and TNFα in the CSF of several ethnic populations of people living with HIV (PLH) , 48–50. Cognitive impairment in HIV-infected patients may be caused directly by the action of the HIV virus or its well-known auxiliary proteins . HIV-1 TOur study has several limitations. First, the sample size is small, particularly for the sub-group analyses that we performed. We believe that our sub-group comparisons were sufficiently sized to generate meaningful conclusions; however, our findings should be interpreted with caution. The need to replicate these findings is important. Second, for the purpose of this study we focused on the role of HIV-1 Tat and inflammatory cytokines in the release of sIR from neuronal cells. The presence of glutamate in the CSF and the possible influence of TNF and Tat combination on the secretion of sIR was not measured. Third, we did not adjust for multiple comparisons in our analysis of sIR secretion from neuronal cells. We believe that adjustment was not necessary for two reasons: (1) we conducted planned comparisons that each asked the same basic question, and (2) there was consistency in the results with all comparisons pointing to the same conclusion. Fourth, we only studied a cohort of women with HIV infection since our cohort was designed to study the effects of HIV in women. Further studies to address sex differences are warranted. Fifth, our study design did not consider differentiating the chronic vs. acute effects of HIV-1 Tat. Future studies comparing this effect should be considered.Soluble receptors, including insulin, may be secreted from cells in several forms such as intact or with an associated cell membrane. Recent studies have observed that the latter form may be secreted as a vesicle as an exosome , 60. In Our study provides evidence that the activation of the TNFα receptor plays an important role in the secretion of sIR from neuronal cells. Both TNFα and HIV-1 Tat regulate the secretion of sIR from neuronal cells. Further studies will investigate whether the effects of HIV-1 Tat on sIR secretion are mediated by neuronal TNFα being released to the extracellular medium.YG, RM-D, JV, AN, and VW were responsible for the conception and design of the study. YG, RM-D, AD-N, JM-Á, JS-P, LH, AN, and VW contributed to the data acquisition. RS, YG, RM-D, AN, and VW were involved in the analysis and interpretation of data. YG, RM-D, and VW drafted the first version of the article. All authors revised it critically for important intellectual content. All authors gave final approval of the version to be submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.